While wogonin has been known to have cytotoxicity against various cancer cells, its bioavailability and cytotoxicity are low due to its low water solubility. Therefore, wogonin-loaded solid lipid nanoparticles were fabricated using a hot-melted evaporation technique. The highest solubility of wogonin was observed in stearic acid. Hence, wogonin-loaded solid lipid nanoparticles were composed of stearic acid as the lipid matrix. The physicochemical properties of the wogonin-loaded solid lipid nanoparticles were evaluated by dynamic laser scattering and scanning electron microscopy. The wogonin-loaded solid lipid nanoparticles exhibited sustained and controlled release up to 72 h. In addition, it was observed that the wogonin-loaded solid lipid nanoparticles exhibited enhanced cytotoxicity and inhibited poly (ADP-ribose) polymerase in MCF-7 breast cancer cells. Overall, the results indicate that wogonin-loaded solid lipid nanoparticles could be an efficient delivery system for the treatment of breast cancer. Scutellaria baicalensis and possesses anticancer properties in a wide range of human cancer cell lines by inducing apoptosis in vitro [Wogonin  is one of the active compounds of the flavonoids isolated from the radix of in vitro ,2,3,4. Ain vitro . In addiin vitro ,10.Solid lipid nanoparticles (SLNs) are nano-sized particle carriers that consist of a biocompatible and biodegradable solid lipid matrix that remains in the solid state in the human body ,12,13,14The aim of this study was to develop and optimize the wogonin-loaded solid lipid nanoparticle (W-SLN). W-SLNs were able to encapsulate and release wogonin efficiently to MCF-7 breast cancer cells. It was highlighted that wogonin from W-SLNs can be released in a controlled manner to achieve a sustained cytotoxic effect on MCF-7 cells. w/w), respectively. Based on these results, stearic acid was chosen as the solid lipid.The solubility of wogonin in the lipid matrix is a key point for determining drug loading and encapsulation efficiency in SLN. For this purpose, the solubility of wogonin in three different solid lipids was determined to maximize the drug loading. As seen in W-SLNs were prepared by a hot sonication method. Next, physicochemical properties such as particle size, polydispersity index, and encapsulation efficiency (E.E) were determined. The formulation variations in terms of loading amount of wogonin and their effect on the properties of W-SLNs are listed in A drug release study of the wogonin solution and W-SLN was conducted . Wogoninp < 0.05). In other words, W-SLN exhibited time-dependent inhibition efficiency up to 72 h while the cell viability of MCF-7 treated with the wogonin solution was recovered over time. As shown in In order to investigate the mechanism of wogonin-induced inhibition on cell proliferation, each cell cycle was monitored using fluorescence-activated cell sorting (FACS) to measure the cell distribution. The cell cycle after exposure to the wogonin solution or W-SLN was measured by flow cytometry at 24, 48, and 72 h. As shown in For qualitative cellular uptake of SLN, Nile red was encapsulated—instead of wogonin—in SLN. The fluorescence images were taken by confocal microscopy. Similar to the quantitative cellular uptake studies, Nile red-loaded SLN exhibited higher fluorescence than the other formulations in MCF-7 cells. In addition, there was no obvious difference in fluorescence between free Nile red and blank SLN with free Nile red .To determine poly (ADP-ribose) polymerase (PARP) concentrations in MCF-7 cells after exposure to the wogonin solution or W-SLN, a Western blot assay was conducted after cells were treated with free wogonin or W-SLN. The results of the Western blot assay for PARP expression levels are shown in In this study, wogonin-loaded solid lipid nanoparticles were prepared using a hot sonication method. Although wogonin is a natural compound with known therapeutic effects on a diverse range of cancer cells, its clinical use is limited by its low bioavailability and fast elimination . SLN hasThe in vitro release studies were conducted in PBS (pH 6.8). The result shows that wogonin in W-SLN exhibited a bi-phase release pattern. It is clear that the encapsulation of wogonin in SLN allowed for sustained and controlled release. The release mechanism of wogonin from W-SLN can be explained by the distribution of wogonin in the lipid matrix. In other words, some wogonin that localized in the surface is related to an initial release burst and the wogonin encapsulated in the lipid core can be released in a sustained manner by slow diffusion through the SLN ,30,31. IFurthermore, W-SLN showed, by cell inhibition studies, sustained cytotoxicity against cells. The lipid matrix (stearic acid) of the nanoparticles showed no toxicity on MCF-7 cells ,36. W-SLThe cellular uptake of different formulations was evaluated in MCF-7 cells. The free wogonin solution exhibited poor cellular uptake of wogonin, while W-SLN was easily taken up by the cells, strongly indicating the SLN resulted in a more extensive and prolonged exposure of wogonin to the cells relative to the wogonin solution. In order to confirm the successful cellular uptake of SLN formulations, Nile red was introduced into SLN instead of wogonin. Since wogonin does not possess autofluorescence, Nile red was chosen as a dye because it is also a hydrophobic molecule with a similar molecular weight. It is highlighted that W-SLN exhibited the highest fluorescence over time, while Nile red solution and blank SLN with Nile red did not exhibit obvious fluorescence over time. This result correlated well with the enhanced cytotoxicity and cellular uptake of wogonin from W-SLN. The cell cycle is a major event that leads to cell cleavage and replication. In addition, regulating the cell cycle is an important step in the survival of single cells. The cell cycle is divided into four sequential phases: G1, S, G2, and M. Cancer cells are mainly characterized by the disruption of cell cycle regulation, resulting in dysregulation of cell proliferation. The regulation of the cell cycle is a crucial process in cell survival. For example, regulating the G1/S and G2/M checkpoint enables control of the progression of the cell cycle and, consequently, it can be a strategy for the elimination of cancer cells. Wogonin induces cycle arrest at the G0/G1 phase in diverse types of cancer cells ,41. As sApoptosis is the programmed cell death regulated by different enzymes and genes. PARP, a DNA repair enzyme activated by DNA damage, has been used as a biochemical marker of apoptosis . TherefoWogonin hydrate, Dynasan 118, and mannitol were obtained from Sigma-Aldrich . Stearic acid and GMS were purchased from Samchun Chemical Co., Ltd. . Lecithin and poloxamer 188 were obtained from Junsei  and BASF , respectively. Dialysis membrane (Membra-Cel) was purchased from Viskase, Inc. . To choose an ideal solid matrix, the solubility of wogonin was measured by melting in stearic acid, GMS, and Dynasan 118. In brief, each lipid (2 g) was melted 10 °C above the melting point of the lipid in a water bath and wogonin (10 mg) was added to the melted lipid until saturation was achieved . The samPreparation of wogonin-loaded SLN was performed by a modified hot sonication technique . FirstlyEthanol (1 mL) was added to the SLN solution (0.5 mL) in a hot water bath (80 °C) to dissolve the lipid matrix of SLN. After 10 min, the mixture was kept in the fridge (4 °C) to precipitate the lipid matrix. The clear supernatant was filtered using a syringe filter (0.22 µm) to remove precipitated lipid and drug. The filtered solution (20 μL) was analyzed using HPLC system  with a mobile phase (75% methanol and 25% ammonium acetate buffer (pH 4.0) distilled water) at wavelength 270 nm with a 1 mL/min flow rate. The column used was a Hedera ODS-2 column . Limit of detection (LOD) and limit of quantitation (LOQ) were 20 ng/mL and 50 ng/mL, respectively. Coefficient of determination (R2) was 0.9991 within the concentration range of 0.05–500 μg/mL. E.E was calculated as follows:E.E (%) = measured amount of drug in SLN/weight of the used drug × 100.The freeze-dried SLN (3 mg) was added to distilled water (3 mL) under sonication for 10 s. Then, dynamic laser scattering analyzer  was used to measure particle size. Zeta potential of SLN was measured by a Zetasizer Nano Z . The in vitro release profile of wogonin from wogonin solution and W-SLN was determined using a dialysis membrane (MWCO 7k). Wogonin solution was prepared by dissolving wogonin in ethanol. Wogonin solution or W-SLN (20 µg of wogonin) was immersed in PBS . At pre-determined time points, 1 mL of release medium was replaced with fresh medium (1 mL). Wogonin concentration was analyzed by HPLC as mentioned in the 2 at 37 °C.MCF-7 cells were obtained from the American Type Culture Collection . The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin in a humidified atmosphere of 5% CO4 cells/mL) was seeded into 96-well plates and incubated for 24 h. Wogonin solution or W-SLN was added and incubated for 24, 48, and 72 h. After that, the medium was discarded and the MTT solution  was added. After 4 h, the medium was replaced with DMSO (200 µL). A microplate reader  was used to measure the absorbance at 570 nm.The cytotoxicity of the wogonin solution and W-SLN against MCF-7 cells was measured using the MTT  assay. Briefly, 200 µL of MCF-7 cells (5 × 106 cells per well. After incubation (24 h), the cells were treated with different formulations for 4 or 8 h. After that, the medium containing the formulation was discarded and the cells were washed with cold PBS three times. The cells, then, were lysed using 1% Triton X-100 (400 μL) and the cell lysate was used to determine the total cell protein amount by the BCA assay. The concentration of wogonin in the cell lysate was measured by HPLC as described above.For the quantitative uptake study, MCF-7 cells were seeded in a 6-well plate with a density of 1 × 106 cells per well in growth medium (1 mL) and incubated for 24 h to allow them to attach to the dish. Then, the cells were treated with the different formulations in growth medium. At predetermined time points (4 or 8 h), the cells were washed three times with cold PBS. Then, cells were stained with 1 μg/mL DAPI in PBS for 3 min and washed twice with PBS. The cells were observed directly under the CLSM.For the qualitative uptake study, Nile red was encapsulated in SLN. The cells were incubated on a glass-based dish  and observed by confocal laser scanning microscopy . In the glass-based dish, the MCF-7 cells were seeded at a density of 1 × 10After treatment for 24, 48, and 72 h, the cells were collected by centrifuging at 1500 rpm and washed with PBS. Then, PI and ribonuclease were added to stain the cells. Flow cytometer  was used to measure the cell cycle.g at 4 °C. The soluble fractions were processed for SDS-PAGE at 100 V for 120 min and immunoblotting onto a PVDF membrane was performed using a PowerPacTM HC . PARP (116 kDa) or β-actin (42 kDa) was detected using PARP antibody  or β-actin antibody , followed by a secondary antibody IgG-HRP: sc-2004 (1:4000 dilution).After exposure to different formulations for 24, 48, or 72 h, the formulation was discarded and MCF-7 cells were washed thrice with PBS. The cells were lysed in a mammalian protein extraction reagent (MPER). The cell lysates were centrifuged for 15 min at 15,000 t-test was used to compare two different groups of samples. A p-value < 0.05 was considered significant. The Student’s Wogonin was successfully encapsulated in solid lipid nanoparticles using stearic acid as the lipid matrix. The release rate of wogonin from SLN was slower than that of pure wogonin. Furthermore, the W-SLN may be a promising and potent carrier for the delivery of wogonin against the breast cancer cells. Future work will involve the synergistic effects of conventional anticancer drugs and wogonin in the SLN system as wogonin has a unique cytotoxic mechanism.


The VLBT-BSANPs were obtained under optimum conditions, with a mean particle size (MPS) of 155.4 nm and a zeta potential (ZP) of −32.97 mV at a pH value of 5.4. Drug loading efficiency (DLE) and drug entrapment efficiency (DEE) of this obtained drug were approximately 45.6% and 90.6%, respectively.Vinorelbine tartrate (VLBT), as a kind of high hydrophilic and temperature-induced degradation drug, was prepared into nanoparticles by a desolvation procedure. Bovine serum albumin (BSA), as a drug carrier, was stabilized by chemical cross-linking with glutaraldehyde. Firstly, the optimization process of preparing VLBT-loaded BSA nanoparticles (VLBT-BSANPs) was accomplished using response surface methodology (RSM) by desolvation. Then VLBT-BSANPs were conjugated with folate, namely Fa-BSANPs-VLBT. Hence targeting drug carrier delivery system loading VLBT was produced. In this study, the characteristics of the nanoparticles, such as the amount of folate conjugation, surface morphology, surface chemistry, physical status of VLBT in Fa-BSANPs-VLBT, stability of Fa-BSANPs-VLBT with mannitol and  Their ce (VLBT)  developethe vein . Thus, ie market . The adve market . However® IV and Navelbine® Oral, the present study was carried out to discover an ideal carrier or a novel strategy for VLB so as to reduce the severe venous irritation, enhance the anti-tumor activity and improve patient tolerance. It has been reported that many kinds of drug delivery systems loading VLB have been developed. For example, much attention has been focused on the study of liposome-encapsulated VLB [In view of the disadvantages of both Navelbineated VLB ,21,22,23ated VLB ,25. HoweBefore our study, Zu and colleagues prepared vinblastine sulfate (VBLS)-loaded folate conjugated bovine serum albumin (BSA) nanoparticles . In thisin vivo, their particle size plays a key role in determining body distribution and promoting their access into cancer cells after the folate has targeted cancer tissues. A statistical approach using a central composite design was used to optimize particle size and maximize DLE. The four-factor and five-level central composite design (CCD) matrix and experimental results obtained are presented in X1, X2 and X4), and finally, the best-fitted model equations are shown as;When the nanoparticles are injected X1 and X4 were found to have significant effects on the MPS. So these variables were chosen to plot the response surface for the MPS while holding X2 and X3 at central points, 0.55 mg/mL and 3.25 mL/min, respectively. As can be seen from X1) was increased. According to the ANVOA, the ratio of BSA concentration and VLBT concentration (X2) had pronounced effects on DLE. As shown in X2).According to the ANVOA, ® software and based on the criterion of desirability. The optimized formulation was achieved with 3.29 mg/mL BSA, 3mg/mL VLBT, 2.84 mL/min of ethanol rate, and 3.44 fold of the ethanol amount . The VLBT-BSANPs were obtained under optimum conditions, with a MPS of 155.4 nm and a ZP of −32.97 mV. The DLE and DEE of this obtained drug were approximately up to 45.6% and 90.6%, respectively. Thereafter, all of the following experiments were conducted using VLBT-BSANPs produced by this optimized formulation.Nanoparticles with the MPS of 100 to 200 nm had an enhanced permeability and retention (EPR) effect, which allowed the preferred accumulation of drug-loaded nanoparticles within tumors . MoreoveN-hydroxysuccinimide ester of folate (NHS-folate) was conjugated with 1 mg BSANPs according to the corresponding standard curve, namely y = 2877x + 645.9, R2 = 0.9995 . In Karsten Ulbricha’s research, folate-conjugated human serum albumin (HSA) nanoparticles showed favorable targeting efficiency for tumor cells when 7.40 ± 0.90 μg folate was bound per mg HSANPs [Due to the low accuracy of UV spectrophotometry, high performance liquid chromatography (HPLC) was used to achieve a quantitative test on the folate content associated with the VLBT-BSANPs. The result indicated that 5.67 μg g HSANPs . Moreoveg HSANPs .In order to examine and compare the surface characteristics of the nanoparticles resulting from each step in preparing the optimal formulation of Fa-BSANPs-VLBT, they were prepared and scanned using scanning electron microscopy (SEM). In The VLBT-BSANPs b were evThe aggregation phenomenon directly caused that the MPS of Fa-BSANPs-VLBT with mannitol increased to about 300 nm  measured−1 due to the COOH bond, which proved that VLBT had been entirely wrapped up inside the BSA. The phenomenon that VLBT was entirely wrapped up inside the BSA made VLBT circulate in blood for long periods of time and improved the stability of VLBT in circulation.The X-ray diffraction (XRD) results for raw VLBT, VLBT-BSANPs, Fa-BSANPs-VLBT and Fa-BSANPs-VLBT with mannitol are shown in The thermogravimetric analyzer (TGA) results, used to examine the thermal property of raw and VLBT-loaded nanoparticles, are shown in Moreover, the TGA result of VLBT-loaded nanoparticles indicated that they had a higher weight loss percentage compared with the raw VLBT at the same temperature. This was possibly due to the fact that VLBT-loaded nanoparticles with small MPS had a higher specific surface area, which was in favor of cellular uptake of cancer cells, and hence had a higher specific surface energy, which subsequently lead to an easier vaporization and earlier decomposition energy.in vitro drug release experiments over 140 h, the results of which are shown in y = −58.35ex/5.56)(− − 20.47ex/33.01)(− + 79.55 (R2 = 0.9934). Meanwhile, the release of Fa-BSANPs-VLBT with mannitol corresponded with the Higuchi equation of y = −33.36ex/8.86)(− − 40.59ex/21.76)(− + 76.32 (R2 = 0.9967). The observed release curve indicated that the drug carrier system had a sustained release property that facilitated the application of nanoparticles for the delivery of anticancer drugs.The Fa-BSANPs-VLBT powder with mannitol was reconstituted to carry out The MPS of Fa-BSANPs-VLBT measured by DLS was bigger than that observed by SEM owing to aggregation. Moreover, the conjugation of the folate with the BSANPs and lyophilization of Fa-BSANPs-VLBT increased the MPS of VLBT-BSANPs from155.4 nm to about 300 nm, and the ZP of VLBT-BSANPs changed from −32 mV to about −17 mV  when theN-hydroxysuccinimide , 1,3-dicyclohexyl-carbodiimide , Dimethyl Sulphoxide , ethanol (purity > 99.5%) and the other reagents were all of analytical grade.Folate, bovine serum albumin, trypsin, phosphate-buffered saline (pH 7.4), BSA trypsin and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were all obtained from Sigma Aldrich . The VLBT was kindly provided by Hisun Pharmaceutical Co. Ltd. . Acetonitrile and ethanol were of high performance liquid chromatography grade. Three grams of folate were dissolved in 60 mL dimethyl sulfoxide (DMSO) containing 1.5 mL triethylamine, and then 2.82 g DCC and 1.56 g NHS were added to the solution under stirring. The mixture was stirred for 12 h at room temperature. The insoluble dicyclohexyl urea was removed by filtration, and then the filtrate was poured into ice-cold anhydrous ether solution containing 30% acetone, followed by centrifugation at 3000 g for 5 min at 4 °C and washed twice using ether. Finally, NHS-folate, a delicate light yellow solid powder, was obtained by drying at room temperature .3 buffer. The VLBT was loaded mainly through electrostatic interaction between positive charges of the drug and negative charges of the BSANPs, when the pH value of the solution was higher than the isoelectric point of BSA. Huang and colleagues have reported this mechanism of loading drugs [The VLBT-BSANPs were prepared as previously described using a desolvation procedure. In the preliminary trials, we found that if VLBT was dissolved in purified water, the formation of BSANPs as drug carriers was unstable with precipitation. Thus the pH for the VLBT water solution was adjusted to about six with a NaHCOng drugs . 3, the buffer solution together with BSA, and subsequently the desolvation agent of ethanol were added dropwise into the aqueous BSA and VLBT solution under magnetic stirring with a peristaltic pump TI/62/20  at room temperature. The opalescent suspension was formed spontaneously and was further examined as nanoparticles. After the desolvation process, 0.25% glutaraldehyde in water (v/v) solution was added to crosslink the amino groups of the VLBT-BSANPs. Crosslinking was initiated under stirring of the colloidal suspension over a time period of 24 h. Ethanol in the prepared sample was eliminated by evaporation using a rotary evaporator R201BL  at 40 °C, and then the VLBT-BSANPs were redispersed to the original volume with a NaHCO3 buffer solution. The redispersion process was performed in an ultrasonication bath TI-H-5  for about 3 min. This redispersion solution was centrifuged  to separate the free VLBT. After each centrifugation, the residue was redispersed to the original volume, with deionized water, using a mechanical stirrer. This purification process was repeated three times. Finally, the supernatant from each centrifugation was collected to determine the DEE and DLE. The nanoparticles settled were obtained correspondingly before lyophilization.As illustrated in X1), the ratio of BSA concentration and VLBT concentration , the rate of adding ethanol , and the ratio of ethanol and VLBT solution  as the independent variables (Y1) and MPS (Y2) of the resulting VLBT-BSANPs were used as response variables. Design-Expert® Version 7.0.0 software  was applied to generate and evaluate the statistical experimental design.The above-described VLBT-BSANPs preparation procedure was optimized by a four-factor, five-level central composite design (CCD), with BSA concentration  and added slowly to the VLBT-BSANPs solution while stirring. The reaction was maintained for 120 min at room temperature. Then the mixture was centrifuged at 10,000 rpm for 20 min three times and washed twice with NaHCO3 buffer solution, followed by freeze-drying. As shown in The lyophilization of Fa-BSANPs-VLBT prepared in deionized water was performed using a Gamma 2-20 apparatus  to make sure the physical stability of the ultimate product along with the addition of mannitol as lyoprotectant . The prepared Fa-BSANPs-VLBT was redispersed in 4 mL deionized water in 8 mL glass vials after purification and centrifugation. The mixture was prefrozen in the refrigerator (−20 °C) for 12 h and subsequently lyophilized at −40 °C for 24 h, followed by a secondary drying phase of 12 h at 20 °C.18 column,  at ambient temperature. The mobile phase consisted of 50 mM phosphate buffer solution containing 8% methanol, which was adjusted to pH 6.3 with concentrated KOH solution. The system was run isocratically at a flow rate of 1 mL/min, and NHS-folate was detected at 254 nm. Briefly, the Fa-BSANPs-VLBT was digested by trypsin (50 g/mg BSA). Then the resultant tryptic hydrolysis of the Fa-BSANPs-VLBT was examined by HPLC. The amount of NHS-folate conjugated with VLBT-BSANPs was determined by high performance liquid chromatography (HPLC) using a Diamonsil C18 column. The mobile phase was a mixture of acetonitrile and 50 mM phosphate buffer solution containing 1% triethylamine , running at a flow rate of 1 mL/min. Detection was accomplished at 268 nm. DEE and DLE were calculated using equations as previously reported by Park and colleagues [The free VLBT in the supernatant was examined by HPLC. The total VLBT was the feed VLBT during preparation process of Fa-BSANPs-VLBT. In Formula 4, Fa-BSANPs-VLBT referred to the lyophilized powder of Fa-BSANPs-VLBT. A Waters HPLC , consisting of a Waters 600 Controller equipped with a Waters 717 plus autosampler, and a Waters 2487 UV detector, were used. The samples were chromatographed at 25 °C by injecting 10 μL sample into a Diamonsil Clleagues .(3)(4)The obtained nanoparticles were analyzed for their MPS and ZP by dynamic light scattering (DLS) equipment  with a He-Ne laser  as light source. The measurement was performed in triplicate at 25 °C and with angle detection at 90° in 4 mm diameter cells with a field strength of 10 V/cm. The samples were prepared by diluting the nanoparticle suspension with deionized water. The data were obtained from the average of several measurements.The morphology of the samples was determined by using scanning electron microscope . The total number of particles scanned was not less than 100. Samples were coated by gold before examination (cathode dispersion).−1 at a resolution of 4 cm−1.The samples were diluted with KBr mixing powder at 1% and pressed to obtain self-supporting disks, separately. The Fourier transform infrared (FTIR) spectrum was obtained by IRAffinity-1  and recorded in the wave number range of 4000–500 cmThe X-ray diffraction (XRD) patterns were collected in transmission using an X-ray diffractometer with a rotating anode . The scanning rate was 5°/min, and the diffraction angle (2θ) was recorded at 3°–90° .The samples were irradiated using a Cu target tube at 30 mA and 40 kV. 2). Analysis was performed for 5.0 mg samples at a temperature heating rate of 10 °C/min and a temperature range of 30–225°C.Thermal analysis was carried out using differential scanning calorimetry  for the samples prepared in this experiment. All thermal analyses were performed in an inert atmosphere . During the course of testing, the samples weighing 2–3 mg were heated at a fixed heating rate of 10 °C /min from 35 to 500 °C under a nitrogen purge.In vitro drug release study was carried out in phosphate buffered saline  at 37 °C. Fa-BSANPs-VLBT suspension  and raw VLBT  was placed, respectively, in treated dialysis bag , and then the dialysis bag was immersed in a 250 mL beaker containing 200 mL release buffer. The beaker was then placed on a magnetic stirring apparatus, which was in an incubator shaker at 37 °C, and the speed of the paddle was adjusted to 100 rpm. Five milliliters of sample were withdrawn at predetermined time intervals and equivalent amount of fresh release buffer was replaced. The drug content in the withdrawn samples was estimated by HPLC using conditions as described above. The accumulative release percentage was calculated using the following equations:The iC represents the VLBT concentration of each sample withdrawn at predetermined time intervals, i'C represents the increase of the VLBT concentration during each time interval, V represents the volume of the release buffer, V0 represents the volume of each withdrawn sample, M is the weight of VLBT loaded in the Fa-BSANPs-VLBT, and iQ is the accumulative release percentage at a predetermined time point. The drug release studies were carried out in triplicate for each of the samples. The drug release from optimized formulations was compared to raw VLBT, and mathematical models were applied to study the release kinetics.The stability of the Fa-BSANPs-VLBT study was tested with the help of DLS equipment . The ultimate product along with the addition of mannitol as lyoprotectant  were analyzed for their MPS and ZP when the pH value was 5.4 at predetermined time intervals . The measurement conditions were determined as described above.In this study, RSM was used to optimize the procedure for the preparation of VLBT-BSANPs. The results indicated that BSA concentration and the ratio of ethanol and the VLBT solution (v/v) played significant roles in determining the VLBT-BSANPs particle size, and the ratio of BSA concentration and VLBT concentration had a significant influence on DLE. The VLBT-BSANPs were obtained under optimum conditions, with a MPS of 155.4 nm and a ZP of −32.97 mV. DLE and DEE of the obtained drug were approximately up to 45.6% and 90.6%, respectively. The obtained spherically shaped Fa-BSANPs-VLBT also had a small average diameter, and their DLE and DEE were considerable. in vivo and reduction of drug toxic action [in vivo properties of Fa-BSANPs-VLBT targeting drug delivery systems will be evaluated in further research. In addition, BSA will be substituted by human serum albumin in further experiments to avoid a possible immunologic response in vivo. The nanoparticles showed a small particle size, a roughly spherical surface morphology, a high zeta potential, a high specific surface area, a sustained release property and the feature of being entirely wrapped up inside BSA. All these characterizations contributed to a good drug delivery system. Moreover, the stability experiment results showed that Fa-BSANPs-VLBT with mannitol was a suitable drug delivery system. Folate-conjugated albumin nanoparticles had a lot of advantages in the areas of cytotoxicity, anti-tumor activity c action ,36,37,38


Haemophilus influenzae (NTHi) protein D conjugate vaccine (PHiD-CV) efficacy in a double-blind randomized trial  against various diseases, including acute otitis media (AOM). Here, we provide further analyses. In the Panamanian subset, 7,359 children were randomized (1:1) to receive PHiD-CV or control vaccine at age 2/4/6 and 15–18 months. Of these, 2,000 had nasopharyngeal swabs collected. AOM cases were captured when parents sought medical attention for children with AOM symptoms; surveillance was enhanced approximately 2 y into the study through regular telephone calls or home visits by study personnel, who advised parents to visit the clinic if their child had AOM symptoms. Mean follow-up was 31.4 months. Clinical AOM (C-AOM) cases were assessed by physicians and confirmed by otorhinolaryngologists. Middle ear fluid samples, taken from children with C-AOM after specific informed consent, and nasopharyngeal samples were cultured for pathogen identification. For 7,359 children, 2,574 suspected AOM cases were assessed by a primary healthcare physician; 649 cases were C-AOM cases as per protocol definition. From the 503 MEF samples collected, 158 resulted in a positive culture. In the intent-to-treat cohort , PHiD-CV showed VE against first C-AOM  and bacterial (B-AOM) episodes  in children <24 months, which declined thereafter with age. Pre-booster VE against C-AOM was 30.7% ; post-booster, −6.7% . PHiD-CV VE was 17.7%  against moderate and 32.7%  against severe C-AOM. VE against vaccine-serotype pneumococcal NPC was 31.2%  3 months post-booster, and 25.6%  across all visits. NTHi colonization rates were low and no significant reduction was observed. PHiD-CV showed efficacy against C-AOM and B-AOM in children younger than 24 months, and reduced vaccine-serotype NPC.We previously reported 10-valent pneumococcal non-typeable  Streptococcus pneumoniae and non-typeable Haemophilus influenzae (NTHi) are the main bacterial pathogens responsible for pediatric AOM in Argentina, Colombia, Mexico, Venezuela, Costa Rica and Chile, in line with observations from other regions worldwide.Acute otitis media (AOM) is one of the most common bacterial diseases in young children, with a peak of incidence at the age of 6–18 months.197-conjugate vaccine ..Synflorix™, GSK Vaccines, Rixensart, Belgium), a recently published randomized controlled trial (RCT) in Finland reported a trend towards reduced AOM.197-conjugate vaccine , no RCTs assessing AOM have yet been reported, although impact on AOM has been assessed in post-marketing studies.Until recently, efficacy/effectiveness against AOM was evaluated in double-blind randomized studies for only one of the 3 licensed pneumococcal conjugate vaccines (PCVs), the 7-valent CRMIn 7vCRM studies, the overall AOM incidence was reduced by 6–7% in children vaccinated with 7vCRM, although more important reductions were reported for more severe AOM cases and for children with recurrent AOM episodes.Previously, we reported efficacy against AOM in the COMPAS trial for PHiD-CV, together with efficacy against other disease endpoints. Our double-blind randomized clinical trial, conducted in Latin America, showed PHiD-CV efficacy of 19.0%  against first episodes of C-AOM, and 69.9%  against C-AOM caused by vaccine pneumococcal serotypes in the intent-to-treat analysis.S. pneumoniae, H. influenzae and other bacterial pathogens, evaluated in a subset of children from the COMPAS trial.Furthermore, the impact of PHiD-CV on nasopharyngeal carriage is relevant, since vaccines directed against the major bacterial pathogens causing AOM could prevent the disease by at least 2 mechanisms: a direct one due to antibodies exuded into the MEF, as well as indirectly via impact on nasopharyngeal colonization of those pathogens. Reductions in nasopharyngeal carriage of vaccine pneumococcal serotypes were seen with 7vCRM, with a significant decrease in vaccine-serotype carriage after the 7vCRM booster  in a study in the US,The numbers of children included in the intent-to-treat and per-protocol cohorts are shown in As previously presented, 7,359 children were randomized to receive PHiD-CV or control; 7,214 children were included in the intent-to-treat cohort for AOM efficacy and 5,989 in the per-protocol cohort for AOM efficacy .6Mean efficacy follow-up for AOM was 28.2 months  for the per-protocol cohort and 31.4 months  for the intent-to-treat cohort.Of the 2,000 children  enrolled in this subset of the AOM efficacy cohort, 1,921 children (955 in the PHiD-CV group and 966 in the Control group) were included in the intent-to-treat cohort for analysis of carriage . The numIn total, 2,574 AOM cases were assessed by a primary healthcare physician (PHCP); of these, 2,215 cases (86%) were seen by an ear, nose and throat (ENT) specialist. Out of the 1,813 AOM cases assessed by PHCP with at least 2 reported symptoms, 649 cases (36%) after ENT assessment were considered as C-AOM cases as per protocol definition . In addition, of a total of 503 MEF collected, 345 samples (69%) turned out to be documented as culture-negative cases.From an analytical point of view, a new AOM episode was defined when ≥ 30 d elapsed from the start of a previous episode. During the intent-to-treat follow-up, 637 C-AOM episodes (294 in the PHiD-CV group and 343 in the Control group), occurring in 562 children (8% of the enrolled children), were confirmed . The incS. pneumoniae, 45 with H. influenzae, 9 with S. aureus, 8 with Group A streptococcus, 2 with M. catarrhalis and 1 with other bacteria (Enterobacter cloacae), with 4 of the 117 episodes having both S. pneumoniae and H. influenzae bacteria; 112 of these B-AOM episodes were first episodes .Point estimates for vaccine efficacy (VE) against first C-AOM were positive for the younger age groups: 26.9% for the 2–11 months group, 19.7% for the 12–23 months group, and 24.0% for both groups pooled together (i.e. children < 24 months of age), with the lower limit of the 95% CI being above zero for the 2–11 months and <24 months age groups. However, VE declined in older children, with point estimates of −10.5% in the 24–35 months age group and −33.8% in the ≥ 36 months age group .In a post-hoc analysis by sex, the incidence of, and VE against C-AOM seemed to be higher in boys .Although none of the VE estimates for the different AOM endpoints analyzed reached statistical significance, the consistent trend across the different endpoints suggests a positive effect, with VE point estimates of 17.7% against moderate and 32.7% for severe AOM in the analysis based on complete symptom reporting, and 12.2% against mild AOM episodes when assessing episodes with information on at least one AOM symptom .VE against first B-AOM episodes was 33.6%   against first episodes of pneumococcal AOM, and 69.9%  against vaccine-serotype AOM , 19F  and 23F  . The prevalence of each of the 7 other vaccine serotypes was below 1.0% in the PHiD-CV group and below 1.5% each in the Control group at each time point. There was no evidence of an impact on carriage of vaccine-related serotypes; VE against carriage of any vaccine-related pneumococcal serotype ranged from −27.9% to 15.8% at the different timepoints, and was 2.0% across all time points. The prevalence of vaccine-related serotypes ranged from 2.4% to 5.2% for serotype 6A, from 1.1% to 2.2% for serotype 19A, and from 0.9% to 1.9% for serotype 23A ; no higher non-vaccine, non-vaccine-related serotype colonization rate was observed in the PHiD-CV group versus the Control group up to 1 month post-booster  at each time point (H. influenzae detected were NTHi. The presented data only include results from samples confirmed as positive for H. influenzae or NTHi after differentiation from H. haemolyticus by polymerase chain reaction (PCR) assay; at least 93% of the cultured NTHi samples were confirmed to be NTHi after PCR differentiation. We did not observe a significant reduction of nasopharyngeal NTHi carriage at any point, with the highest VE being 27.0%  at 3 months following booster vaccination; VE across all time points was 7.4%  , and H. influenzae which had a markedly lower prevalence (<6%), we also assessed the colonization with other bacteria including S. aureus, S. pyogenes and M. catarrhalis. Of these other bacteria, S. aureus was the most commonly isolated pathogen in both groups (approximately 8%). The colonization rate at 9 months post-booster was 6.5%  in the PHiD-CV group and 6.3%  in the Control group . No evidence of an increase in rate of S. aureus carriage in the PHiD-CV group vs. the Control group during the study was found. The percentage of nasopharyngeal samples with S. pyogenes or M. catarrhalis colonization was ≤0.8% in both groups at each time point.In addition to H. influenzae  tended to be lower in the PHiD-CV group than in the Control group. The overall rates of S. aureus, S. pyogenes and M. catarrhalis acquisition were within the same ranges in the PHiD-CV and Control group.Across all sampling time points, vaccination with PHiD-CV was associated with reduced acquisition of vaccine pneumococcal serotypes compared with the control vaccine . Acquisition rates for vaccine-related serotypes or other pneumococcal serotypes were within the same ranges in the PHiD-CV and Control groups. Acquisition of In this study in more than 7,000 children in Panama, PHiD-CV showed VE against AOM in children younger than 24 months. However, VE declined with age. Our data also suggested a trend towards efficacy against AOM with different levels of severity. Because most cases of AOM were first episodes according to our study definition, the trial can be considered as a study of first AOM episodes. Additionally, a consistent trend for reduction of vaccine-serotype nasopharyngeal carriage was observed in PHiD-CV recipients compared with the Control group.We previously reported VE against first C-AOM episodes of 16.1%  in the per-protocol analysis and 19.0%  in the intent-to-treat analysis , the total number of reported AOM episodes was lower than could be expected based on incidences in other countries, indicating that our study probably underestimates the true AOM burden. However, the fact that most AOM episodes (≥ 90%) were mild or moderate in severity indicates that there was no substantial bias towards detecting only severe AOM episodes.Various factors might have contributed to the low incidence of AOM in this study, including antibiotic use, low frequencies of smoking among parents, and high breastfeeding rates.,VE against C-AOM was mainly driven by VE against moderate and severe episodes (even though CIs span 0); no VE against mild episodes was observed when assessing only episodes with complete positive and negative reporting, on all 5 symptoms, though an indication of such VE was seen when incompletely documented cases were included. We suggest it is reasonable to assume that for cases with missing reports for one or more symptoms, the physician did not document the symptom(s) because they were not present during examination. This could explain why nearly 30% of mild C-AOM cases could not be included in the analysis of severity with complete reporting (vs. 13% of moderate and 6% of severe episodes).We note that the proportion of MEF samples with culture positivity was low, and the bacterial etiology of AOM could only be confirmed for a limited number of episodes. This may have been due to antibiotic treatment before MEF collection. We did not find evidence for laboratory issues or problems with transport of the samples that could explain the low culture positivity rates.In the present study, PHiD-CV VE against both C-AOM and B-AOM was observed in children below 24 months of age, but appeared to decrease after the second year of life. The fact that no (C-AOM) or limited (B-AOM) evidence of VE was observed following the booster vaccination was unexpected, since robust immune responses were induced by the booster dose in the present study, as described previously.Streptococcus pyogenes, Moraxella catarrhalis and Staphylococcus aureus as bacterial agents that cause AOM.In the current study, VE against World Health Organization-defined consolidated community-acquired pneumonia (CAP) remained above 20% among children aged 12–24 months and 24–36 months.In PMS studies, decreases in AOM incidence have been observed after introduction of PCV13 and PHiD-CV. When compared to the 7vCRM period, a 19% reduction in OM was observed after introduction of PCV13 in the UK,VE against AOM episodes caused by vaccine pneumococcal serotypes was 70% in the intent-to-treat analysis and 67% in the per-protocol analysis, which was in line with the results of a previous trial conducted in Finland with 7vCRMS. aureus was generally consistent between groups. Nevertheless, an increase in carriage of non-vaccine, non-vaccine-related serotypes was observed after the PHiD-CV booster dose, suggesting that some pneumococcal serotype replacement may occur in the niche created by vaccine-type nasopharyngeal carriage reduction. This rise could not easily be attributed to any specific non-vaccine serotype, as carriage rates for individual non-vaccine, non-vaccine-related serotypes did not exceed 2.3% at any time point (data not shown). The examination of individual serotypes showed that the observed increase in the PHiD-CV group was caused by additional isolates for a number of serotypes. This tendency suggests that caution should be exercised in assuming that serotype replacement may not expand over long-term periods.The nasopharyngeal colonization rate with vaccine pneumococcal serotypes was consistently lower after vaccination with PHiD-CV in comparison with the control vaccine in the present study, while acquisition of any vaccine-related serotype, any other non-vaccine, non-vaccine-related serotype, or Nevertheless, the fact that trends for elevated carriage of non-vaccine pneumococcal serotypes were only seen at 3 and 9 months post-booster, long after the decrease in vaccine-type carriage was apparent, contrasts with the results of randomized clinical trials and post-marketing studies showing that 7vCRM vaccination tended to be associated with serotype replacement concurrent with or only slightly delayed after vaccine-type decreases.A dynamic model of pneumococcal infection predicted that vaccines with relatively small effectiveness against colonization by vaccine serotypes could have a major impact on the incidence of invasive pneumococcal disease (IPD) caused by those serotypes in the target population.The reductions of vaccine pneumococcal serotype carriage seen in this study will likely have clinical significance, as reductions with other PCVs have been closely associated with substantial herd protection of unvaccinated individuals.S. pneumoniae,While an association has been made between pneumococcal disease and nasopharyngeal carriage of It is difficult to conclude on the clinical relevance of the limited (or no) effect of vaccination on overall NTHi carriage, especially if only a pathogenic subset of NTHi is actually responsible for clinical AOM.This is the first study reporting simultaneous assessment of the effects of PHiD-CV on both carriage and AOM with assessment of bacterial etiology. However, this study has a number of limitations. First, the study was limited by the low number of reported AOM and of bacteriologically confirmed cases. Moreover, the results of the descriptive analyses presented in this manuscript should be interpreted with caution because there was no adjustment for multiplicity and no pre-defined success criteria were set. Finally, the interpretation of the descriptive analyses of VE per age subgroup and before vs. after booster dose was limited by the difficulty of separating the effect of age from the effect of the booster dose.In conclusion, PHiD-CV showed efficacy against AOM in children aged less than 24 months, who are the most vulnerable population for AOM and its consequences. Positive VE estimates were observed against a range of AOM severity categories up to the booster dose administration, indicating that the results from this trial are likely relevant to the commonly seen clinical range of AOM cases and that PHiD-CV vaccination may have a considerable impact on public health. The decreases in AOM were accompanied by significant decreases in vaccine-serotype nasopharyngeal colonization with only a small, delayed tendency for increases in colonization with non-vaccine-type pneumococci and no evidence of changes in carriage of other pathogens. These findings are consistent with emerging evidence of herd protection against disease in unvaccinated age groups in countries using PHiD-CV.COMPAS was a phase III, double-blind, randomized controlled study conducted in Argentina, Colombia and Panama . Its primary objective was to demonstrate PHiD-CV efficacy against first episodes of likely bacterial community-acquired pneumonia (B-CAP) in infants and young children. The study objectives and key efficacy results for CAP, AOM and IPD have already been reported.Synflorix™) and diphtheria–tetanus–acellular pertussis–hepatitis B–inactivated poliovirus–Haemophilus influenzae type b vaccine (Infanrix hexa™) at 2, 4, and 6 months of age followed by one dose of PHiD-CV at 15–18 months of age, while the control group received hepatitis B vaccine (Engerix-B™) and diphtheria–tetanus–acellular pertussis–inactivated poliovirus–Haemophilus influenzae type b vaccine  followed by one dose of hepatitis A vaccine (Havrix™).In Panama, the study was conducted between August 2007 and July 2011 in 16 centers. A total of 7,359 children were randomized (1:1 ratio) to receive either PHiD-CV (PHiD-CV group) or control vaccines (Control group) at 2, 4, 6 and 15–18 months of age, as described previously.Written informed consent was obtained from children's parents or guardians. The study was conducted in accordance with Good Clinical Practice, all applicable regulatory requirements and the Declaration of Helsinki, except for the deviations that were described previously,The methodology for the surveillance of AOM was described previously,If AOM was suspected by a PHCP, the child was referred to an ENT specialist involved in the trial. The diagnosis of C-AOM required at least 2 clinical symptoms  which had started within the previous 5 d and were reported by the PHCP; and either altered visual appearance of the tympanic membrane  or the presence of middle ear effusion as observed by the ENT specialist.MEF sampling through tympanocentesis was proposed to children with a C-AOM that was confirmed by an ENT specialist. Initially, if the severity according to the OS-8 scaleH. influenzae, S. pneumoniae, Staphylococcus aureus, Streptococcus pyogenes or Moraxella catarrhalis) was identified in the MEF sample using standard microbiological culture methods .An C-AOM was considered of bacterial etiology (B-AOM) if a bacterial pathogen recognized as causative of AOM . If an AOM case was reported within 30 d from the start date of a previous episode, it was considered as the same clinical episode. A new B-AOM episode was defined whenever a different bacterial pathogen was isolated at the latter visit. First AOM episodes were defined as the first episode reported for this child in the course of the study.et al. was used to assess the severity of each suspected AOM case.The clinical/otologic scale developed by Dagan The impact of PHiD-CV on nasopharyngeal carriage was assessed in a subset of the AOM efficacy cohort, consisting of 2,000 children (carriage subset). Nasopharyngeal swabs were taken at 6 time points: at 7 months of age (1 month after primary vaccination), 12–15 months of age (approximately 6–9 months after primary vaccination), before booster vaccination (15–18 months of age), and 1, 3 and 9 months after the booster dose .Nasopharyngeal swabs were collected by study personnel using pediatric rayon-tipped swabs with flexible aluminum shaft, although non-flexible swabs were also used in approximately 200 samples because of temporary unavailability of flexible swabs at some centers. Microbiological procedures are detailed in the text of Supplemental Digital Content 7.The determination of the sample size and study power, the definitions of the study cohorts, and the calculation method for VE were described previouslyS. pneumoniae and NTHi, were descriptive with no adjustment for multiplicity. No pre-defined success criteria were fixed but from a descriptive point of view, conclusion on statistical significance of VE was based on a positive lower limit of its non-adjusted 95% CI and statistically significant differences between groups were defined based on non-overlapping 95% CI boundaries around incidence rates. However, no strict statistical conclusions can be made in these kinds of exploratory analyses; the focus is on trends.PHiD-CV VE against first C-AOM episodes occurring in the per-protocol cohort as of 2 weeks after dose 3 was considered significant if the primary study objective to demonstrate VE against first B-CAP episodes was significant and the one-sided P-value for VE against C-AOM was < 0.025; the results of these confirmatory objectives were reported in the primary manuscript.et al, as described above.PHiD-CV VE against AOM was further characterized descriptively  by age , before vs. after booster vaccination , and by severity . AOM severity was categorized according to the 5 symptoms used in the clinical/otologic scale developed by Dagan For the analysis according to AOM severity, different levels of severity for the same AOM episode  were counted as individual endpoints and analyzed as separate AOM episodes . As a consequence, the sum of all mild, moderate and severe episodes could be higher than the total number of AOM cases counted when not considering the severity. Cases of the same severity level occurring within 30 d  were counted as 1 episode. The same methodology was applied for the analysis of AOM episodes reported before and after the booster dose. For the analysis by age, the total number of reported AOM episodes was counted considering the appropriate cohort and follow-up, and the 30-day rule was applied for the analysis of all episodes. Episodes were then subdivided per defined age range according to the age of children at the time of the considered episode. The sum of episodes in the various categories was thus equal to the total number of episodes.S. pneumoniae, H. influenzae or other bacterial pathogens at each swab time point, as well as the frequency of acquisition of new bacteria or serotypes were calculated with non-adjusted 95% CIs. A new acquisition at a particular visit was defined when a child's nasopharyngeal swab became positive for a specific bacterium or serotype after being negative at the previous visit for that specific bacterium or serotype.Percentages of children with nasopharyngeal swabs positive for In the intent-to-treat cohort, PHiD-CV VE against nasopharyngeal carriage of bacterial pathogens was estimated as 1 minus the relative risk. Statistical significance of VE was assessed based on a lower limit of the 95% CI around VE >0 with no adjustment for multiplicity.Prevenar/Prevnar and Prevenar 13/Prevnar 13 are trademarks of Wyeth Pharmaceuticals Inc., a subsidiary of Pfizer Inc. Synflorix, Infanrix, Infanrix hexa, Engerix-B, and Havrix are trademarks of the GSK group of companies.


A submaximal constant-speed test to measure the running economy (RE) and a 5-km running trial on an outdoor track to establish pacing strategy and performance were also done. During the 5-km running trial, the rating of perceived exertion (RPE) and time to cover the 5-km trial (T5) were registered. After the training period, there were significant improvements in the HIIT group of ∼7 and 5% for RE (P=0.012) and PTS (P=0.019), respectively. There was no significant difference between the groups for VO2max (P=0.495) or onset of blood lactate accumulation (P=0.101). No difference was found in the parameters measured during the 5-km trial before the training period between HIIT and CON (P>0.05). These findings suggest that 4 weeks of HIIT can improve some traditional physiological variables related to endurance performance (RE and PTS), but it does not alter the perception of effort, pacing strategy, or overall performance during a 5-km running trial.This study analyzed the influence of a 4-week high-intensity interval training on the pacing strategy adopted by runners during a 5-km running trial. Sixteen male recreational long-distance runners were randomly assigned to a control group  or a high-intensity interval training group . The HIIT group performed high-intensity interval-training twice per week, while the CON group maintained their regular training program. Before and after the training period, the runners performed an incremental exercise test to exhaustion to measure the onset of blood lactate accumulation, maximal oxygen uptake (VO It has been widely recognized that during recreational and official athletic events the running intensity is always self-selected by athletes –3. The m-shaped speed curve during a 10-km running race compared with their counterparts. In addition, high-performance athletes ran the first 1200 m of a 10-km race at a speed faster than the average speed of the entire race and above their PTS, while a low-performance group started the race with a less aggressive pacing strategy and slightly below the OBLA speed .Therefore, the main purpose of this study was to analyze the influence of a 4-week HIIT program on pacing strategy during a 5-km running trial. Our hypothesis was that the HIIT program might improve physiological variables related to running pacing strategy , resulting in an altered U2/d2, as proposed by Hopkins (2max) before the beginning of the study and were instructed to maintain this aerobic training schedule during the experimental period. The participants’ running training volume was reported as the mean distance covered per week  to determine time to exhaustion at this speed (TLim), which was used for individualizing the HIIT program . After a 3-min warm-up at 8 km/h, the speed was increased by 1 km/h every three minutes until exhaustion. The treadmill was set at a gradient of 1% to simulate physiological demand during outdoor running . Each sttal test . The vVOntration . PTS wasThe RE was determined on a motor-driven treadmill . Participants performed a standardized warm-up, consisting of 5 min of running at 8 km/h followed by a 5-min passive recovery. Thereafter, they performed a constant-speed running test at 12 km/h for 10 min in order to measure the RE. During the entire test the oxygen uptake was obtained breath-by-breath. RE was defined by averaging the oxygen uptake values during the last 30 s.2max was immediately adjusted after the warm-up and the participants ran until they could no longer maintain the required speed. The test began with the participant’s feet on the moving belt and hands on the handrail. The TLim was measured using a manual stopwatch and defined as the moment that the participant released the handrail (about 2 s) until he grasped it again . The participants received strong verbal encouragement to continue as long as possible.The participants in the HIIT group performed the same warm-up routine adopted during the RE test. The vVOad libitum during the entire event. The runners performed a 10-min, warm-up consisting of a free-paced run, followed by 5 min of light stretching. The RPE was reported by participants every 1000 m using the Borg 15-point scale (T5) were registered by a GPS every 400 m . The pattern of data collecting for RPE , T5, and HRT5  was according with a previous study carried out by Lima-Silva et al. . The running speed during the HIIT was monitored by a GPS (GPS Forerunner¯ 305). The training sessions were individually supervised to control the training loads. Over this 4-week training period, the CON group was instructed to maintain their previous endurance training routine.The HIIT group performed a high-intensity interval training program twice weekly (separated by 48 h) for 4 weeks in addition to their normal endurance training. The athletes were instructed to perform their regular endurance training on different days to those of the HIIT sessions. In order to equal the training load between the training regimes, there was a reduction of ∼10% of the total endurance training volume  in the HIIT group. A standardized warm-up consisting of a 5-min run at 9 km/h followed by light lower-limb stretching exercises was performed before each training session. Because Tcription , in the t-test. Significance was accepted at P<0.05. All statistical analyses were performed using the software package Statistica 8 .Data normality was confirmed using the Shapiro-Wilk test. Two-way analysis of variance (group × time) was used to compare the physiological and performance variables. In order to mitigate the impact of inter-individual data variability, physiological variables are also reported as percentage of change from pre-training period . Comparison between groups for percent of changes (%) after the experimental period was performed using unpaired Lim used for prescription of HIIT was 265±67 s. No statistical difference was observed in the endurance training volume (reported as the mean weekly covered distance) between before  and after  the completion of the study (P>0.05), indicating that training load was equal between training regimes.All of the participants in the HIIT group completed over 85% of the scheduled training sessions. The mean value of T=0.012) and PTS (P=0.019) when compared with the CON group. There was no significant difference between the groups for VO2max (P=0.495) and OBLA (P=0.101). >0.05), group (P>0.05), or interaction (P>0.05) for all measured variables.>0.05). There were no significant main effects for time, group, nor interaction effects for T5, HRT5, and RPET5 (P>0.05). >0.05).The main objective of the present study was to investigate the effects of the addition of a 4-week HIIT program on the pacing strategy adopted by long-distance runners during a 5-km running trial. The main findings were that the HIIT program improved physiological variables related to endurance performance , but these changes were not accompanied by modifications in pacing strategy or overall performance.2max . Smith et al. (2max, 5.2% in vVO2max, and a non-significant improvement in 5-km running performance. In addition, Billat et al. (The improvement of only ∼2.5% in 5-km running performance detected in the present study is in agreement with others that reported small enhancement in overall running performance after a 4-week HIIT program ,15,16. Ft et al.  found noIt is important to acknowledge some of the limitations of the present study. First, our participants were recreational long-distance runners who had only low-intensity continuous aerobic training experience. Thus, caution should be taken in extrapolating these findings to highly-trained athletes who frequently perform HIIT training sessions. Second, the athletes individually performed the 5-km running trial, while during official competitive running races, they compete in a head-to-head manner. Previous findings have suggested that the presence of other competitors would alter the pacing strategy, inducing to a more aggressive and faster start and improving overall performance . This coIn conclusion, the results of the present study showed that the addition of 4 weeks of HIIT produced relevant gains on the PTS and RE, but without changes in RPE, pacing strategy, and overall performance. These findings suggest that improved aerobic power and lower energy cost during submaximal running were not sufficient to alter the perceived effort behavior during a 5-km running trial, resulting in a similar pacing strategy to that used before the training period.


Introducing multifunctional groups and inorganic material imparts xylan-based hydrogels with excellent properties, such as responsiveness to pH, temperature, light, and external magnetic field. In this work, a composite hydrogel was synthesized by introducing acid treated carbon nanotubes (AT-CNTs) into the maleic anhydride modified xylan grafted with poly(N-isopropylacrylamide) (MAX-g-PNIPAM) hydrogels network. It was found that the addition of AT-CNTs affected the MAX-g-PNIPAM hydrogel structure, the swelling ratio and mechanical properties, and imparted the hydrogel with new properties of electrical conductivity and near infrared region (NIR) photothermal conversion. AT-CNTs could reinforce the mechanical properties of MAX-g-PNIPAM hydrogels, being up to 83 kPa for the compressive strength when the amount was 11 wt %, which was eight times than that of PNIPAM hydrogel and four times than that of MAX-g-PNIPAM hydrogel. The electroconductibility was enhanced by the increase of AT-CNTs amounts. Meanwhile, the composite hydrogel also exhibited multiple shape memory and NIR photothermal conversion properties, and water temperature was increased from 26 °C to 56 °C within 8 min under the NIR irradiation. Thus, the AT-CNTs reinforced MAX-g-PNIPAM hydrogel possessed promising multifunctional properties, which offered many potential applications in the fields of biosensors, thermal-arrest technology, and drug-controlled release. Hydrogel is a gel-like material that consists of water-soluble polymers cross-linked physically or chemically. It swells by absorbing a great quantity of water rather than being dissolved in water ,2. Recen3+-CMH) were produced by the two-step process, which possessed the high strength and great elasticity [In recent years, natural-occurring polymers have been intensively studied for fabricating intelligent hydrogels in drug release, adsorption and many other fields  due to tasticity . The PAMasticity , which dβ-d-1,4-xyloside bond with a variety of side groups, which as the major hemicellulose is in grasses and in dicot secondary cell walls [Xylan is the second-dominating polysaccharide after the cellulose and is linked by ll walls . There all walls ,15,16. Fll walls . MAX coull walls . The MAXll walls . TherefoCurrently, nanotechnology and nanocomposites could create opportunities for fabricating nanomaterials. Carbon nanotubes (CNTs) are a kind of one-dimensional quantum material with nanoscale at radial dimension and micron grade at the axial dimension. CNTs have unique electrical characteristics that are beneficial for endowing composite materials with electrical conductivity ,21,22. FN,N-methylene-bis-acrylamide (MBA) as the cross-linker and acid treated CNTs (AT-CNTs) as the reinforcment. The influence of AT-CNTs was discussed on the hydrogel structure, swelling ratio, mechanical strength, conductivity, and photothermal conversion properties. The objective of this paper was to fabricating new intelligent MAX-based hydrogels possessing multifunctional properties such as high mechanical strength, electrical conductive, thermo-sensitive properties as well as the shape memory. These hydrogels were prepared by the cross-linking polymerization of MAX with NIPAm using MAX was prepared according to our previous work (DS = 0.64) . CNTs we2 g of CNTs were dispersed in 80 mL mixed acids in which the volume ratio of concentrated sulfuric acid to concentrated nitric acid was 3:1 and the mixture was treated by sonication for 8 h. After the treatment, the mixture was precipitated for 24 h followed by four times dilution with deionized water and dialyzed in water for seven days until pH reached to neutral. AT-CNTs were finally obtained after lyophilization .In this work, NIPAM (1.3–2.0 g) and a specific amount of MAX (0–0.7 g) were added in deionized water and stirred in the ice bath. Then different contents of AT-CNTs  were added into the mixture solution. The solution was sonicated and stirred. After the homogenous mixture was obtained, 0.05 g APS, 0.05 g MBA were added. After adding 6 μL of TMEDA to the mixture, the obtained homogenous solution was poured into molds to proceed further polymerization at 5 °C and finally the hydrogel was formed. The hydrogel was then soaked in deionized water to remove the impurities. The preparation conditions of the MAX-g-PNIPAM/AT-CNTs hydrogel are shown in The changes of bonds in CNTs and AT-CNTs were characterized using Raman Spectrometer  equipped with a Spectra-Physics Ar: Kr laser source for an excitation at 532 nm.The Fourier transform infrared spectroscopy (FT-IR) spectroscopies of CNTs, AT-CNTs, MAX, xylan and hydrogels were determined by Vertex 33 spectrophotometer . All samples were dehydrated in an oven at 50 °C before testing, and then the finely ground samples were mixed with KBr pressed into a plate for the measurement.The morphology of AT-CNTs was observed using a transmission electron microscopy (TEM) . AT-CNTs were dispersed and diluted in ethanol, followed by ultrasonic treatment before drying on a copper wire.The X-ray diffraction (XRD) patterns of CNTs, AT-CNTs, MAX, and hydrogels were collected by D8 ADVANCE X-ray diffractometer  at a speed of 2° per minute and a range of 2θ = 10–50°. The hydrogel was lyophilized and ground into a powder prior to the test.The morphology of hydrogels was observed by scanning electron microscopy (SEM)  with an accelerating voltage of 10 kV. The hydrogels were dehydrated by vacuum freeze-drying before testing.A simultaneous thermal analyzer  under a nitrogen flow of 20 mL/min was applied to determine the thermogravimetric property of the hydrogels. 10 mg dried hydrogel sample was ground to powder and heated from 25 °C to 700 °C at a 10 °C/min heating rate in an open alumina crucible.The compressive strength of the hydrogel was measured by Tensile Compression Material Testing Machine . The circular hydrogel was swelled at room temperature to achieve swelling equilibrium before testing.d and Ws represent the weights of the dried hydrogel and the hydrogel at swelling equilibrium state, respectively.Swelling ratio was tested by the immersion of the dried hydrogels in deionized water until their weight became constant. The hydrogels were then removed from the water and weighed on balance after removing the excess water by gently tapping the hydrogel sample with filter paper. The swelling ratio was calculated using the following equation:The low critical solution transition (LCST) of the swollen hydrogels was measured by a differential scanning calorimeter (DSC) with a heating rate of 5 °C/min under a nitrogen atmosphere (25.0 mL/min nitrogen flow rate) and a thermal analysis temperature range from 15 °C to 55 °C. The hydrogel was swelled at room temperature to achieve swelling equilibrium before testing.The electrical conductivity of the hydrogels was measured at room temperature while using the FT-340 four-point probe method . The hydrogel was swelled at room temperature to achieve swelling equilibrium before testing.The hydrogel was placed in a cuvette containing deionized water which were divided into experimental group (E) and control group (C). The near-infrared (NIR) laser source was then directed at the experimental group and irradiated at a distance of 5 cm. The NIR laser source has a power of 2 W and a wavelength of 808 nm. After radiation of different times by a NIR laser, the water temperature in the cuvette was immediately measured with a temperature probe.The rectangular MAX-g-PNIPAM/AT-CNTs hydrogel (gel-13) was placed in a 10% NaCl solution at room temperature to obtain a definite shape, and then placed in deionized water to observe the shape changes.2 atoms in the carbocyclic or long chain. The relative intensity (ID/IG) reflects the degree of disorder and defect density of the sample, and the large the ratio could lead to the high the sample disorder and defect density [D/IG ratio of AT-CNTs was higher than that of CNTs, indicating that the ordered graphite structure in the CNTs was destroyed and replaced by the functionalized AT-CNTs [The changes of the bonds in CNTs and AT-CNTs were determined by Raman spectra and illustrated in  density . The cor AT-CNTs .−1. The absorption band at 3425 cm−1 is owing to the stretching vibration of -OH groups, while the adsorption bands at 2925 and 2864 cm−1 represent the stretching vibrations of aromatic and aliphatic C-H, respectively [−1 and the symmetric shear vibration of C-H is at 1384 cm−1. However, besides those bands, there are also two bands at 1263 and 1124 cm−1 on the AT-CNTs spectra that are related to the stretching vibration of phenolic C-O and alcoholic C-O, respectively. This implied that carboxyl and hydroxyl groups were successfully introduced to the surface of CNTs after acid treatment [FTIR spectra in ectively . After areatment .The TEM micrographs of AT-CNTs were exhibited in d-spacing resembles the one of pristine graphite [XRD patterns of CNTs and AT-CNTs were shown in graphite .−1 [−1 is for the stretching of -OH groups, and the C-H stretching vibration band occurs at 2912 cm−1. The adsorption bands at 1465 and 1043 cm−1 are the stretching vibration bands of -C-H and -C-O-C-, respectively. Arabinose units and β-glucoside linkage are represented by the adsorption bands at 982 cm−1 and 897 cm−1. The absorption band at 1736 cm−1 in the spectrum of MAX is the characteristic absorption band of ester group (C=O), which is indicative of the successful modification of xylan by maleic anhydride [FTIR spectra of MAX, AT-CNTs, and MAX-g-PNIPAM/AT-CNTs dried hydrogels were illustrated in −1 . A broad−1 is derived from the C=O of NIPAM, and the asymmetric adsorption band of COO− appears at 1458 cm−1 [−1. In addition, C-H stretching vibration band of MAX is observed at 2933 cm−1, and the asymmetric adsorption band of COO− of NIPAM appears at 1461 cm−1. In the spectrum of MAX-g-PNIPAM/AT-CNTs (gel-9), the characteristic bands of MAX and NIPAM are observed and the phenolic C-O stretching vibration and the alcoholic C-O stretching vibration of AT-CNTs are present at 1277 cm−1 and 1131 cm−1, respectively [In FTIR spectrum of PNIPAM (gel-1), the stretching vibration band at 1643 cm458 cm−1 . In the The swelling behaviors and DSC curves of hydrogels were illustrated in 2O, which needed more energy to be destroyed [The estroyed . The equilibrium swelling ratio of the hydrogels as the functions of MAX amounts in hydrogels was shown in The effects of AT-CNTs addition on the equilibrium swelling ratio was exhibited in The thermal stability of PNIPAM hydrogel (gel-1), MAX-g-PNIPAM hydrogel (gel-5) and MAX-g-PNIPAM/AT-CNTs hydrogel (gel-10) were showed in The same phenomena were seen also from the results of DTG b. From tThe mechanical tests, e.g., compressive strength as well as Young’s modulus of hydrogels were shown in The compressive strength and Young’s modulus of the hydrogel increased with an increase of the MAX amount in MAX-g-PNIPAM hydrogel b. When tThe addition of MAX and AT-CNTs further increased the mechanical properties of MAX-g-PNIPAM hydrogels in It was found that AT-CNTs started to play an important role for the mechanical properties of hydrogels d. Both cMorphologies of the hydrogels were determined by SEM and a homogeneous network structure was observed for PNIPAM hydrogels gel-1, a,d, MAX-−5 S/cm. It was mentioned that the increasing of CNTs amounts resulted in the improvement of the electrical conductivity [The electrical conductivity of the hydrogels was measured and displayed in uctivity . The difThe photothermal conversion of hydrogels was studied in As shown in −5 S/cm. Meanwhile, after irradiating by NIR, the temperature in cuvette with MAX-g-PNIPAM/AT-CNTs hydrogel increased to 55 °C after 8 min of irradiation, indicating that the hydrogel displayed the ability of photothermal conversion. Moreover, hydrogels exhibited multiple shape memory property. The obtained CNTs-reinforced MAX-g-PNIPAM hydrogel would have potential applications in the fields of biosensors, thermal-arrest technology and drug controlled release.In this work, AT-CNTs reinforced MAX-g-PNIPAM hydrogel was successfully synthesized with multifunctional properties, such as electrical conductivity, photothermal conversion ability, and shape memory. The mechanical properties of the hydrogel were sharply increased by the addition of AT-CNTs. When the amount of AT-CNTs addition was 11%, the compressive stress of hydrogel reached to 83 kPa, and the conductivity reached to 20 × 10


K mean, pachymetry, or ECD were found between the two groups at the visit after 6 months of follow-up . At 6 months postoperatively, 2 eyes lost one or more lines of visual acuity in the LASIK-ACXL group, whereas all LASIK-only treated eyes had a stable CDVA. In vivo confocal microscopy showed a decrease of keratocyte density and appearance of honeycomb-like structures in the anterior residual stroma bed; the changes were similar but more pronounced following LASIK-only. None of the cases in both groups developed signs of significant keratitis, regression, or ectasia during the 6-month follow-up. LASIK-ACXL can effectively correct refractive error in patients with myopia, with no significant complications during 6-month follow-up, indicating stability and morphologic change similar to those with LASIK-only treatment.This was a prospective controlled clinical trial. 48 myopia patients (96 eyes) were included in this study. After LASIK, accelerated corneal collagen cross-linking (ACXL) was used for myopia treatment. During 6-month follow-up, the results of LASIK-ACXL treatment were studied and compared to the LASIK-only procedure. The results showed that no statistically significant differences in UDVA, CDVA, MRSE,  The priorldwide . Postopeorldwide . The cliThe corneal collagen cross-linking (CXL) procedure increases covalent bonding between or within collagen fibers in the corneal stroma, thus increasing corneal stiffness and improving corneal biomechanical stability . The lonThe aim of this prospective study was to evaluate the efficacy and safety of LASIK-ACXL as compared to LASIK alone at 1, 3, and 6 months postoperatively. Visual acuity, refractive error, corneal morphology, endothelial cell density (ECD), and the changes of corneal microstructure were recorded and compared with those in the LASIK-only treated eyes.96 eyes of 48 consecutive patients who were scheduled for LASIK surgery from March 2015 to January 2016 in the Department of Ophthalmology Refractive Center, Chinese PLA General Hospital , were enrolled in this prospective comparative study. Half of the patients underwent only LASIK treatment (LASIK-only group) and the other half were treated with CXL and LASIK (LASIK-ACXL group). They were randomized into the two groups. All participants signed an informed consent form in accordance with the tenets of the Declaration of Helsinki. This study also received institutional review board approval of Chinese PLA General Hospital, Beijing, China.μm, predicted postoperative residual stroma bed thickness of less than 280 μm, and patients with any other eye disease, external injury history, systemic diseases, or any other history of ocular surgery. Participants were instructed to remove soft contact lenses at least 2 weeks or rigid contact lenses at least 4 weeks before the screening examination.Inclusion criteria included age ≥18 years, preoperative spherical refractive error from −1.00 D to −11.00 D, refractive cylinder <−5.00 D, stable refractive error for more than 2 years, and willingness and ability to comply with postoperative care. Exclusion criteria included central corneal thickness (CCT) less than 480 K (K mean) values were measured using Pentacam.All subjects underwent a systematic ocular examination preoperatively and follow-up visits at 1, 3, and 6 months. The following were assessed at each visit: UDVA, corrected distance visual acuity (CDVA), manifest refraction, cycloplegic refraction, intraocular pressure (IOP), slit-lamp microscopy, fundus examination, corneal topography , corneal tomography , and ECD . The pachymetry and topographic mean Confocal laser scanning microscopy with a retina tomograph  was used to observe the morphologic changes in the corneal stroma. The physician who evaluated the corneal tissue morphology on the images was blinded to which eye had ACXL.μm or 110 μm and a Wavelight EX500 excimer laser  for refractive correction. All laser parameters and laser nomograms for LASIK were applied according to the clinic's typical LASIK protocol.Conventional bilateral LASIK was performed using a Wavelight FS200  for flap creation with thickness of 90 2  over the closed flap, according to manufacturer's recommendations.Patients were prepared using the same process of flap creation as for LASIK-only group. After the ablation of the corneal stroma, the single-use VibeX Xtra riboflavin drops  were instilled onto the exposed stromal bed for 90 seconds. Then balanced salt solution was used to flush remaining riboflavin from the stromal bed, the flap was then repositioned, and the KXL system  was used to apply 90 seconds of continuous light illumination at 30 mW/cmPostoperative follow-up was at 1 day and 1 week after the surgery. Complete ophthalmic evaluations were conducted at 1, 3, and 6 months postoperatively. Postoperative regimens were prescribed for both eyes as follows: 0.1% fluorometholone eye drop, four times per day, and sodium hyaluronate eye drop, four times per day.t-test. Independent two-sample t-tests were used to compare data between the two groups. If the data were not normally distributed, the Wilcoxon rank-sum test was performed. P values less than 0.05 were considered as statistically significant.Statistical analysis was performed using SPSS 17.0 . The Kolmogorov-Smirnov test was used to check for normal distribution of quantitative data, provided here as the mean ± standard deviation (SD). The significance of the change from preoperation to postoperation was evaluated using the paired P = 0.383) or gender (P = 0.306) distribution between the two groups. The parameters and data before operation and at 1, 3, and 6 months after operation are shown in At 1, 3, and 6 months, 96 eyes were observed. The LASIK-ACXL group included 25 males and 23 females with a mean age of 24.63 ± 3.85 years (range: 18–33 years). The LASIK-only group included 20 males and 28 females aged 18–34 years, with mean age of 25.33 ± 4.06 years. There was no significant difference in age (P < 0.001) and CDVA (P = 0.005) at 1 month after operation. All eyes had emmetropia as the target refraction. At 6 months after operation, the efficacy index (mean postoperative UDVA/mean preoperative CDVA) was 1.02 ± 0.18 in LASIK-ACXL group and 1.06 ± 0.25 in the LASIK-only group. There were no statistically significant differences in UDVA and CDVA between the two groups from preoperation to postoperation except in the UDVA (The safety index (mean postoperative CDVA/mean preoperative CDVA) was 1.09 ± 0.32 at 6 months in the LASIK-ACXL group and 1.15 ± 0.23 in the LASIK-only group. P = 0.074).K mean value between the two groups through the 6-month follow-up. The change of K mean values from 1 month to 6 months after operation in the LASIK-ACXL group was 0.09 ± 1.24 D and in LASIK-only it was 0.13 ± 1.45 D, respectively (P = 0.887). K mean changes in each postoperative follow-up period, demonstrating the stability of the results.There was no statistically significant difference in the topographic P = 0.295; LASIK-only, P = 0.32).ECD in the two groups was not statistically different at 6 months after operation compared to the preoperative data , which increases the length of treatment time (30 min). Both doctors and patients hope to reduce therapy time and increase comfort and improve work efficiency of doctors. Rocha et al. [2; 370 nm wavelength ultraviolet-A light) applied for 3 minutes. The average follow-up duration was 3.5 years (1–4.5 years). The result showed patients had good logMAR vision without complications such as a decrease in ECD, occurrence of corneal ectasia, or refractive regression. The authors concluded that prophylactic ACXL for high-risk LASIK cases appeared to be a safe effective adjunctive treatment for refractive regression and potential ectasia [2; irradiation time: 90 s) in this study. At 6 months of follow-up, we found that LASIK-ACXL is effective and safe in the treatment of myopia, with similar recovery of the LASIK-only treatment method. Morphological changes observed by confocal microscopy, such as a decrease in keratocyte density and appearance of honeycomb-like structures in the anterior residual stromal bed, are similar to those observed after ACXL surgery [It has been demonstrated that CXL can enhance corneal biomechanical properties. According to a long-term follow-up study by Frederik et al. , the maja et al.  put forwa et al. , 17 star ectasia . A more  surgery , 19. ThePostoperative visual recovery is an important index to evaluate the operation efficacy. The efficacy and safety indices were comparable with the studies for the LASIK-only procedure by Chan et al.  and LASIIn this current study, there were no significant differences in refractive error between the LASIK-ACXL and LASIK-only groups postoperatively. Our results are promising and comparable with those published by Tomita et al.  who focuK mean decreased significantly in both LASIK-ACXL and LASIK-only groups and remained stable at follow-up visits at 1, 3, and 6 months, with no statistical differences from 1 to 6 months after operation between the two groups. The steady state of keratometers after surgeries indicated that both surgical methods can effectively improve the visual function of patients and maintain stability of corneal morphology in the early postoperative period, but a longer follow-up period is essential to further verify the stability of these results.2, which is far lower than the threshold to induce corneal endothelial cell loss, iris, crystalline lens, or retinal damage [2, which is also lower than the threshold for corneal endothelial cell damage. Compared with the preoperative values, the ECD of LASIK-ACXL group showed no obvious change after 6 months, pointing out that the combined surgery has no significant effect on postoperative endothelial cells. Many studies [A potential risk of CXL is that it may induce endothelial cell damage. The ultraviolet irradiation energy of the traditional CXL procedure is 5.4 J/cml damage . Althoug studies , 26 haveIn vivo confocal microscopy was used to observe the microstructural changes over time after LASIK. Keratocyte apoptosis and honeycomb-like stromal edema in the anterior residual stroma bed in the early postoperative period were seen after LASIK-ACXL and LASIK-only. It was more pronounced in the LASIK-ACXL group, although the microscopy changes were similar. Tomita et al. [a et al.  also foua et al. , 27. In However, the current study had a few limitations. This was a short-term follow-up study. It is not possible to determine the longitudinal change of LASIK-ACXL treatment for myopia correction. The sample size of this study was relatively small and therefore the results should be interpreted cautiously. Further studies with a larger sample size and a longer term follow-up period are required to corroborate our results.In summary, there were no statistically significant differences in visual acuity, refraction, keratometry, and ECD between the LASIK-ACXL group and the LASIK-only group. Confocal microscopy showed that the keratocyte density decreased and honeycomb-like structures appeared in the anterior residual stroma bed in both groups at one month after operation and they were less pronounced over time during follow-up. Along with the rapid development of ACXL technology, the safety of LASIK surgery, efficacy, and patient selection will further improve.


The purpose of this work is to benchmark RapidPlan against clinical plans for liver Intensity-modulated radiotherapy (IMRT) treatment of patients with special anatomical characteristics, and to investigate the prediction capability of the general model (Model-G) versus our specific model (Model-S).A library consisting of 60 liver cancer patients with IMRT planning was used to set up two models , using the RapidPlan knowledge-based planning system. Model-S consisted of 30 patients with special anatomical characteristics where the distance from planning target volume (PTV) to the right kidney was less than three centimeters and Model-G was configurated using all 60 patients in this library. Knowledge-based IMRT plans were created for the evaluation group formed of 13 patients similar to those included in Model-S by Model-G, Model-S and manually (M), named RPG-plans, RPS-plans and M-plans, respectively. The differences in the dose-volume histograms (DVHs) were compared, not only between RP-plans and their respective M-plans, but also between RPG-plans and RPS-plans.95% of PTV and had greater dose sparing in the right kidney. For the normal liver, RPG-plans delivered similar doses, while RPS-plans delivered a higher dose than M-plans. With respect to RapidPlan models, RPS-plans had better conformity index (CI) values and delivered lower doses to the right kidney V20Gy and maximizing point doses to spinal cord, while delivering higher doses to the normal liver.For all 13 patients, RapidPlan could automatically produce clinically acceptable plans. Comparing RP-plans to M-plans, RP-plans improved VThe study shows that RapidPlan can create high-quality plans, and our specific model can improve the CI of PTV, resulting in more sparing of OAR in IMRT for individual liver cancer patients. Liver cancer is the fifth most common type of cancer and the third leading cause of cancer-related death worldwide. Consequently, liver cancer is an issue that needs to be urgently addressed . IntensiA variety of solutions were studied, to reduce the variability present the back and forth between plan creation and approval –10. AmonIn this study, two models were developed, one general model and one specific model. All the studies mentioned above are based on the general model, which suits a wide variety of patient cases. However, the prediction ability of specific models tailored for specific patient cases, with specific anatomical features, has not been investigated. The aim of this study was (1) to evaluate the accuracy of RapidPlan prediction capability in IMRT  plans for individual liver cancer patients by using model libraries consisting of different total number of plans, with different similarity; and (2) to investigate the prediction capability of the general model vs. our specific model.Liver cancer patients were treated with IMRT from 2015 to 2016, planned based on the Eclipse treatment planning system. For all patients, the IMRT plans were created using 5 non-uniformly distributed coplanar fields  with the same photon beam setting of DVO, including number and beams energy. The prescribed dose was set as 50 Gy in 25 fractions. All plans were normalized to a mean dose of PTV (and isodose 95% was set to the prescribed dose) in order to make plan comparisons valid. OARs planning goals included maximizing point doses to the spinal cord and their planned at-risk volumes (3-mm expansion) below their tolerance dose levels, while lowering the endpoint dose to normal liver tissue and right kidney as much as possible. The PTV dose-volume constraints and OARs dose constraints are shown in Table 3 (range: 15.7-298.3 cm3). No other specific criteria were applied. For Model-S, 30 patients were selected, with the distance from PTV to the right kidney of less than 3 cm. The average target volume was 155.69 ± 77.5 cm3 (range: 30.4-306 cm3). Table The model library consisted of 60 liver cancer patients treated as above. From this library, all 60 patients were selected for Model-G. The average target volume was 147.2 ± 83.6 cmRapidPlan was used to create a knowledge-based model that predicts achievable ranges of DVHs for individual OARs of prospective patients. The model libraries contain all planning CTs, structure sets, and dose distributions of previously treated patients. The model configuration should conceptually consist of multiple kinds of PTV and OAR geometries and alterations in OAR dosimetry, resulting exclusively from geometric alterations.During the training, the system analyzed the patient anatomy and DVHs in the plans using principal component analysis (PCA), and created the final mathematical DVH estimation model. Then, the results of the model training were verified using statistical presentations of the training set. Regression, residual and DVH-plots help in estimating the quality of the model and finding potential outlier values that differ from the average in the training set . The outAn OAR is designated as an outlier when one or more of these metrics lie outside the range of values found in the model. Using this strategy, 14 of 60 patients in Model-G and 6 of 30 patients in Model-S were identified as containing 1 outlier OAR. RapidPlan requires each OAR to be present in at least 20 plans included in the model. We removed statistical outliers from the training set, rather than deleting the whole plan. Table 3 (range: 24-287.5 cm3). OARs typically included the liver, L-PTV , the spinal-cord and their planned at-risk volumes (3-mm expansion). The volumes of the PTV, liver, L-PTV and right kidney, included in the evaluation group, are shown in Table An evaluation group consisting of 13 previous patients, treated in 2016, was used to test the RapidPlan results. Patients in the evaluation group were not included in the RapidPlan model libraries. The evaluation group was similar to that included in Model-S, where the distance from the right kidney to PTV was less than 3 cm. In the evaluation group, the average target volume was 163.1 ± 73.9 cmThe two models were used to generate optimization objectives and automatically optimize treatments for patients in the evaluation group, using the Eclipse treatment planning system. The PTV and OAR objectives are shown in Table 2% - D98%)/Dp) × 100%], where Dp = prescribed dose; a lower HI value indicates that the dose coverage is more homogeneous [PIV2, where TV = target volume, PIV = prescribed isodose volume, and TVPIV = target volume receiving the prescribed dose. The dose coverage and conformity are better when the value of CI is closer to 1. The normal tissue sparing is based on statistical average doses to normal liver , spinal-cord (Dmax), and right kidney . Paired t-tests were performed to determine significant differences (p <  0.05) between RP-plans and their respective M-plans. The target and normal tissue constraints shown in Table Comparisons of the differences in the DVHs, not only between RP-plans and their respective M-plans, but also between RPG-plans and RPS-plans were performed. RapidPlan results were compared based on target dose coverage and normal tissue sparing. The target dose coverage includes: 1) the homogeneity index (HI) calculated for PTV using [HI =  theogeneous : CI = TV the homoAll knowledge-based plans were deemed to conform with the liver IMRT clinical protocol used at our institution, regarding dose-volume constraints. Table 95% of PTV (RPG-plans vs. RPS-plans vs. M-plans: 98.4% vs. 98.4% vs. 97.9%). The D98%, D2%, HI and CI values of PTV were improved, although most differences were not significant. For the OAR, RP-plans had significantly more sparing of the right kidney . For the normal liver, RPG-plans delivered similar doses, while RPS-plans delivered higher doses than those delivered by M-plans . No difference was found between RP-plans and M-plans in the maximum dose to the spinal cord.The comparison of RP-plans to M-plans revealed that RP-plans significantly improved V95%, D98%, D2%, and HI values were very similar, but RPS-plans had better CI values . For OAR doses paring, RPS-plans delivered a lower dose than the maximizing point doses to spinal cord (19.0 Gy vs. 20.7 Gy). Regarding the dose sparing for the right kidney, RPS-plans were better than RPG-plans, especially with V20Gy , although others were very similar. Figure p <  0.05; V20Gy: 20.1% vs. 22.6%, p <  0.05), although all RPS-plans were deemed conform with the liver IMRT clinical protocol. However, for certain individual patients, such as patient 11  and spinal cord, while Model-G delivered lower dose to normal liver tissue. This indicates that the RP-plans are sensitive to the configuration of the model library and the anatomical characteristics of the patient that knowledge-based planning is performed on. The degree of similarity of the cases that make up the model library has a significant effect on the predictive capability of the model.In this study, we evaluated the prediction capability of two models (Model-G & Model-S) in knowledge-based IMRT planning for individual liver cancer patients. Pooled results show that, generally, RapidPlan can improve planning quality and efficiency for liver IMRT, and the prediction ability of the two models with different configurations have a remarkable difference. RP-plans significantly improved the target coverage and the sparing of the right kidney compared to the M-plans. The advantages of some RP-plans compared to the M-plans may be due to challenges in performing an interactive planning of plans that contain many OARs optimally and consistently in a limited number of iterations. Comparison of the two models revealed that Model-S improved the CI and delivered lower dose to the right kidney  the large size of the liver, only considering the cases with short distance between PTV and the right kidney may not meet the requirements for livers located far away from right kidney; (2) Model-G consisted of more cases, which increases geometric heterogeneity. It is conceivable that further improvements could be made and/or some complex tradeoffs should be addressed. Therefore, some parameters may be adjusted during the optimization process. It is worth noting that a review of plans used for RapidPlan models is still required.n = 13). Our study can provide some guidance for clinical applications. Future research will focus on providing optimal allocation of model libraries for individual patients.RapidPlan results depend on a variety of factors and the following two are foremost, the geometry of the region of interest and the quality of the plans contained in the model libraries. To ensure the unity of the variables, we built a specific model, which only considers the geometric distance from the PTV to the right kidney, with no restrictions on the other OAR. In addition, one limitation of the study is the small sample size of the evaluation group (This study shows that RapidPlan can create high quality plans and significantly improve the planning efficiency of IMRT for individual liver cancer patients. Furthermore, these findings demonstrate that the specific model can result in more sparing of OAR, while increasing the conformity index of PTV for liver cancer. Although more systematic studies are needed before a broad clinical application of the proposed methodology, this specific model might be considered as a way to improve the planning quality. Further studies are needed to determine the optimal composition of model libraries, including the relationship between model composition and dosimetry of subsequent plans.